The serum albumins belong to a multigene family of proteins that includes alpha-fetoprotein and human group-specific component, also known as vitamin-D binding protein. The members of this multigene family are typically comprised of relatively large multi-domain proteins, and the serum albumins are the major soluble proteins of the circulatory system and contribute to many vital physiological processes. Serum albumin generally comprises about 50% of the total blood component by dry weight, and as such is responsible for roughly 80% of the maintenance of colloid osmotic blood pressure (see Peters, All About Albumin Biochemistry, Genetics, and Medical Applications, Academic Press, 1996) and is chiefly responsible for controlling the physiological pH of blood (see Figge et al., Lab. Clin. Med. 120:713-719, 1991).
The albumins and their related blood proteins also play an extremely important role in the transport, distribution and metabolism of many endogenous and exogenous ligands in the human body, including a variety of chemically diverse molecules including fatty acids, amino acids, steroids, calcium, metals such as copper and zinc, and various pharmaceutical agents. The albumin family of molecules are generally thought to facilitate transfer many of these ligands across organ-circulatory interfaces such as the liver, intestines, kidneys and the brain, and studies have suggested the existence of an albumin cell surface receptor. See, e.g., Schnitzer et al., P.N.A.S. 85:6773 (1988). The albumins are thus intimately involved in a wide range of circulatory and metabolic functions.
Human serum albumin (HSA) is a protein of about 66,500 kD protein and is comprised of 585 amino acids including at least 17 disulphide bridges. As with many of the members of the albumin family, human serum albumin plays an extremely important role in human physiology and is located in virtually every human tissue and bodily secretion. As indicated above, HSA has an outstanding ability to bind and transport and immense spectrum of ligands throughout the circulatory system including the long-chain fatty acids which are otherwise insoluble in circulating plasma. The atomic structure and particular details regarding the binding affinities of albumin and the specific regions primarily responsible for those binding properties have been previously determined as set forth in references such as He et al., Nature 358:209-215 (1992), Carter et al., Eur. J. Biochem. 226:1049-1052 (1994), Carter & Ho, "Structure of Serum Albumin", in Advances in Protein Chemistry, Volume 45, pages 153-203 (1994), and in co-pending U.S. patent application Ser. No. 08/448,196, filed May 25, 1993, now U.S. Pat. No. 5,780,594, incorporated herein by reference.
In addition to human serum albumin, studies have been made on albumins in a variety of animal species, and it has been determined that over 60% of the amino acid sequences are conserved among the known albumin sequences of many mammals such as bovine, rat and human serum albumin. Moreover, as more and more albumins from other animal species have been sequenced, it has been found that the albumins from a wide range of vertebrate species including sheep, frogs, salmon, mice, pigs and even sea lampreys share a relatively high structural homology, and all share the characteristic repeating pattern of disulphide bridges observed in human serum albumin. In short, all members of the albumin multigene family for which sequences have been determined have good internal sequence homology, thus suggesting that the proteins evolved from a common ancestral protein, and reflecting the vital nature and function of this protein. See, e.g., Carter & Ho, "Structure of Serum Albumin", 1994, referred to above.
Because of the vital role played by albumins, there are literally thousands of applications for serum albumin and its related proteins covering a wide range of physiological conditions, and most often, native serum albumin has been used. However, unlike blood proteins such as hemoglobin, native serum albumins are non-functional as oxygen transport systems, and thus have not been useful in blood replacement systems requiring oxygen transport. As set forth in the recent article by Tsuchida et al., Bioconjugate Chem. 8:534-538 (1997), although the formation of the complex between human serum albumin and hemin has been widely studied, the reduction of hemin to heme and the oxygenation of the heme bound to serum albumin has been difficult or impossible to achieve. For example, Bonaventura et al. (presentation at the 11th Congress of ISABI, Boston, Mass., 1994) preliminarily reported the reversible spectral change of HSA binding a heme derivative in the presence of a large molar excess of axial imidazole upon exposure to dioxin but did not succeed in obtaining a stable oxygen adduct.
In the field of blood replacement products, there are presently two separate and distinct categories of these products, namely those which deal primarily with O.sub.2 transport, such as hemoglobin-based products, and those which are primarily utilized as volume expanders, including products that employ serum albumin. However, supplies with regard to both of these blood replacement products have been severely restricted over the past few years because of numerous concerns with regard to the safety of hemoglobin- and albumin-based products isolated from natural sources because of the concerns that the products will be infected with viral contaminants such as the AIDS virus, the hepatitis-B virus, or a number of other pathogenic microorganisms.
This problem is particularly amplified in the case of albumin since the principal source of this blood protein at present is through isolation from pooled outdated blood which only further increases the risk of infection from viral agents. As a result, the use of serum albumin has been somewhat limited despite the fact that it has been shown to be a very important additive in several different biological applications. For example, in addition to its use as a blood volume expander, albumin has been used as an additive to help preserve organs prior to transplantation. Further, albumin has also been used to promote growth in tissue cultures, and this ability is most likely associated with its role in transport of fatty acids. However, all of these applications are limited because of the inability of albumin to transport oxygen, and because of the problems associated with maintaining an adequate supply of safe and effective albumin.
In addition, even further restrictions on the supply of albumin have occurred because of the awareness of the potential problems of tainted blood, which has led to a steadily decreasing supply of individuals who are even willing to donate blood in the first place. The present awareness of AIDS and other transmittable diseases has caused many people to believe that intravenous blood withdrawal will give rise to the aforementioned disease conditions, and as a result, there has been a declining supply of safe and useful blood replacement products that might have otherwise been obtained from blood donations.
In light of these concerns with natural sources of blood proteins, recent attempts have been made to manufacture and market recombinant sources of blood proteins such as albumin. Currently, several manufacturers produce recombinant albumins, including Delta Biotechnologies, Ltd (see European Pat. App. 201,239), Green Cross Corporation of Japan (see Okabayashi et al., J. Biochem. 110:103-110, 1991), Vepex-Biotechnica, Ltd. of Hungary (see Kalman et al., Nucleic Acids Res. 18:6075-6081, 1990) and Rhone-Poulenc Rorer (see Fleer et al., BioTechnology 9:968-975, 1991). However, these attempts have suffered because the functionality of these blood products are often limited, and in particular there have not been any blood products which can safely and effectively achieve oxygen transport in the manner of the hemoglobin-based products and act as a useful blood volume expander at the same time in the manner of the albumin-based products. In addition, because the present albumin-based products that are available do not transport oxygen, there effectiveness in other applications, such as in organ preservation prior to transplantation, is also quite limited.